Signaling in the nervous system relies on the transfer of chemical signals from one neuron to the next. There are two main classes of these signals: fast neurotransmitters and neuromodulators. Fast neurotransmitters are released from specialized release sites and bind directly to receptors on the opposing post-synaptic membrane to exert an instant change in the membrane potential of the post-synaptic cell. In contrast, neuromodulators are released slowly and diffuse through the extracellular space. They bind to receptors on several cells at once to exert long-lasting changes in a population of neurons. The mechanisms in the secretory pathway for neuromodulators are not well understood. Extensive research from many laboratories on the release of fast neurotransmitters has found that a complex of proteins at the presynaptic membrane, known as the active zone, morphologically docks and functionally primes synaptic vesicles for rapid and precise release. Here I focus on release mechanisms of dopamine, a neuromodulator critical for movement, reward, and emotion. We have recently found that an active zone-like complex of proteins is required for unexpectedly rapid dopamine release. The requirement of a release site strongly suggests that dopamine vesicles are positioned close to their future sites of release and are rendered release ready, reminiscent of the docking and priming of synaptic vesicles. I hypothesize that the specialized, active zone-like release site in dopamine neurons both docks and primes dopamine vesicles to allow for fast exocytosis upon action potential triggering. I will dissect these two processes on a functional and structural level. In aim 1, I will characterize the role of the priming protein, Munc13 in dopamine release. My preliminary data suggests that Munc13 is essential for dopamine release. I will use carbon fiber amperometry, super resolution and confocal microscopy, and mouse genetics to systematically characterize the localization and function of Munc13 in dopamine neurons. In aim 2, I will characterize vesicle docking in dopamine axons and in mutant mice that lack potential docking proteins. To unambiguously identify dopamine terminals, I will employ conditional tagging of vesicles with horseradish peroxidase (HRP) for cell-type identification in electron microscopic images. I will then use serial EM to 3D-reconstruct striatal dopamine axons. In summary, the experiments proposed here will contribute to a novel mechanistic understanding of the dopamine secretory pathway. Our finding that dopamine release occurs rapidly and precisely signals the beginning of a paradigm shift for dopamine transmission. The proposed work expands on dissecting the make- up and function of the rapid exocytotic machinery for dopamine. Precise understanding of dopamine secretion will also provide new insights into how dopamine signaling may break down in neurological disease.